Carbohydrate Research, 66 (1978) 187-194 0 Eisevier Scientific Publishing Company, Amsterdam - Printed in Belgium

STRUCTURAL STUDIES OF ALGINIC ACID, USING A BACTERIAL POLY-or-L-GULURONATE LYASE*f

JONATHAN BOYDAND .~AME.S R. TTJRVEY

School of Physical and Molecuhu Sciences, Uniuersiry CoHege of North Wales, Rangor. Gwyneda LLS7 2UW (Great Britain)

(Received January 27th, 1978; accepted for publication, February 13th, 1978)

ABSTRACT

An extracellular poly-c+L-guluronate lyase from Kkhiella aerogenes degrades those blocks from alginate which contain both mannuronic and guluronic acid residues (poly-MG blocks) to a mixture of oligosaccharides. From an analysis of these products, it is concluded that poly-MG blocks do not have a strictly alternating sequence of the two uranic acid residues. Enzymic degradation of various samples of algal alginate to leave the poly-M blocks intact has shown that these blocks have a uniform chain-length, estimated at 24 residues.

INlXODUCTION

Although it is generally accepted that alginic acid from brown algae consists of a copolymer of three main l&linked structural blocks, poly-/l-D-mannuronic acid (poly-M, l), poly-or-L-guluronic acid (poly-G, 2), and blocks in which the two uranic acids occur together (poly-MG)‘.‘, detailed information is still lacking on two important points. The first of these is the length of the blocks, and the second is the fine structure of poly-MG blocks. The second point has recently been investigated by both chemical3 and enzymic methods4. In a previous paper5, we described the isolation, from KIebsieZla aerogenes type 25, of a poly-a-L-guluronate lyase that degraded poly-G blocks by a random action, to give a mixture of unsaturated oligo- uronides of general structure 3, but had no action on poly-M blocks. The enzyme caused degradation of whole alginate to a mixture of products of high and low molecular weight and had extensive action on poly-MG blocks. We have now examined the products of action of the enzyme on poly-MG blocks, from which some structural features of these blocks can be deduced. The action on whole alginate has also been examin ed, and the large, undegraded fragments have been isolated; from these, the M-block lengths have been estimated.

*Dedicated to Dr. Elizabeth Percival. TStudies of Aiginate-Degrading Enzymes: Part II. For Part I, see Ref. 5.

188 J. BOYD, J. R. TURVBY

1 R’ = C02H.RZ = H 3 n = 0.1.2

2 R’ = H,RZ = CO.+

For convenience in discussing the structures of various products, the following abbreviations will be used: M- and -M- indicate non-reducing-end and 1,4-Iinked /I-o-mannuronic acid residues; -G and -G- indicate reducing-end and l/l-linked a-r_-guluronic acid residues; and A- the unsaturated residue 4, produced at the non- reducing end of a chain by lyase action.

RESULTS AND DWXJSSION

In a previous paper’, we found that the alginolyase gave unsaturated di-, tri-, and tetra-saccharides (3, n = 0, 1,2) from poIy-G, but it was not established whether these were true end-products, or whether further degradation could occur. The action of the enzyme on the oligosaccharides G-G, G-G-G, G-G-G-G, and -A-G-G-G

was investigated. In no case was any appreciable action apparent at an enzyme concentration sufficient to cause extensive degradation of poly-G blocks in less than 1 h. Although not degraded, G-G-G was an inhibitor of enzyme action on poly-G blocks. Table I shows the effect of added trisaccharide 02 the rate of degradation of poly-G blocks, from which it is concluded that the trisaccharide is probably a competitive inhibitor. The products of enzyme action, established previously, there- fore represent true end-products.

TABLE I

INHiBITION OF ENZYME ACTION BY TRIGULURONIC ACID

Substrate cont. (IO- % gdwonare)

Rate of increase in absorbance at 230 nm (IO-“.sec- ‘)

inhibitor cont. (Z(r- ‘M)

0 3 6

0.66 4.60 3.76 2.50 0.99 5.70 4.17 3.25 1.32 6.70 6.17 3.52

Action on poly-MG blocks. - As noted previously5, poly-MG blocks were readily degraded by the enzyme, to give unsaturated oligosaccharides. Examination of the products by t.1.c. suggested that four components were present, each having an appreciable RF value consistent with oligosaccharides containing 2-5 units. Gel filtration on a column of Biogel P-4 established that there was no material excluded

ALGINIC ACID 189

by the gel and enabled four fractions (A-D, in order of elution) to be isolated. The elution volumes of these fractions were consistent with their containing penta-, tetra-, tri-, and d&saccharides, respectively. In p.c., fractions, A, B, and C migrated as discrete bands, but fraction D contained two components, Dr and D, (in approximate ratio 4:l). D, had R,,,, 0.52, which is similar to that (0.53) of the disaccharide, d-G, isolated previously5. Component D, had RGLcA 0.31, suggesting that it might be a saturated disaccharide, since a component havin, (J a similar mobility has been reported6, and identified as the diuronide M-M. It was noted previously that gel atration on Sephadex G-10 separated the disaccharide G-G from salts, but that d-G was eluted from this gel admixed with salts. Accordingly, fraction D was separated into components Da, eluted before salts, and D,, eluted with salts but subsequently desalted on a charcoal column. Fraction & was not detected by absorption at 230 nm, indicating that it did not contain the unsaturated residue 4, but it was detected by the reagent for uranic acids’; fraction D, was detected by both methods. Oxidation of each fraction with periodate and subsequent reaction with thiobarbituric acid, to form* the chromophore with absorption at 550 run, confirmed that fractions A, B, C,

and Dr each contained the unsaturated residue, but that D2 did not. In order to further characterise some of these oligosaccharides, fractions C,

D, , and D, were each lactonised, and the products were reduced with sodium boro- hydride and then hydrolysed with acid5*‘. The resulting mixtures of aldoses and

alditols were examined by electrophoresis in borate buffer, as described previously’. Fraction C gave glucitol, mannose, and non-migrating material; fraction D, gave glucitol and non-migrating material; fraction & gave glucitol and mannose. Fraction

D, , which had a similar R, value to that of d-G, would be expected to give glucitol

and degradation products from the unsaturated unit (non-migrating in borate buffer) if it had this structure. The saturated disaccharide Dz, which should gjve an

alditol (from the reducing end) and an aldose, can, therefore, be assigned the structure

M-G. It must have arisen from the non-reducing end of the poly-MG blocks, which have a relatively short chain-length. The products from fraction C show that this must

have contained the trisaccharide A-M-G, but the fraction might also contain A-G-G,

since, after this treatment, internal G-units give 1,6-anhydro+gulose (non-migrating in borate buffer)_ Examination of fraction C by r3C-n.m.r. spectroscopy suggested

that fraction C was a mixture of these two trisaccharides, and the presence of two components was subsequently confirmed by p.c_ lo. Visual estimation of the intensities

of the zones in p.c. suggested that A-M-G and A-G-G were present in the ratio 4:l. Fractions A and B were not examined further, since it seemed probable that they were mixtures and only small quantities were isolated. The yields of the various oligosaccharides, which were estimated from the areas under the peaks obtained frolm the Biogel P-4 column and as indicated above, are given as molar ratios in Table II.

Some important conclusions regarding both the specificity of the enzyme and the structure of poly-MG blocks can be drawn from the data in Table II. All of the evidence obtained here and previously5 places an L-guluronic acid residue at the reducing end of the oligosaccharides, in agreement with the postulate that the enzyme

190 J. BOYD, J. R. TURVBY

TABLE II

OlKiOSACCHARIDES FROM ENZYME-DEGRADED POLY-MG BLOCK.5

Fraction Oligosaccharide Molar ratio

Dl A-G 8 02 M-G 2

A-M-G 4 C

A-G-G 1

B A-X-X-G” 2 A A-X-X-X-G” 2

“X may be either an M or G residue.

is specific for the L-guluronosyl linkage, and thus Ieads to partial structures for fractions A and B (TabIe II). The sample of poly-MG blocks used in these experiments contained 55% of mannuronic acid and 45% of guluroaic acid. In order to obtain this ratio from the oligosaccharides listed in Table II, it is necessary to assume that some, if not all, of the d-residues must have arisen from 4-linked M-residues in the original blocks. Thus, although the enzyme is specific for the L-guluronosyl linkage, the aglycon residue may be either M or G, both of which give rise to the d-u&t on lysis. An endo-poly-cr-L-guluronate lyase from a pseudomonad has recently been shown to have a similar specificity, although it gives different end-products”. The structure of poly-MG blocks was originally thought to be a strictly altemating sequence of M- and G-residues, with any homopolymeric sequences present only at chain ends”. Simionescu er aL3, on the basis of kinetic studies of the hydrolysis of poly-MG blocks, deduced a more random structure, with -M-M-G- sequences relatively common. Recently, Min et al4 used an enzymic method and concluded that both -M-M-G- and -G-G-M- sequences are present in poly-MG blocks. Our results support this conclusion in that, even if we assume that all of the d-units in Table II arise from M-residues in the original blocks, a minimum of six M-M sequences and one G-G sequence must be present per 53 uranic acid residues. If, as seems probable in view of the isolation of d-G-G, some d-units have arisen from G-residues, the proportion of homopolymeric sequences is greater, and strictly alternating M-G regions may not be the preponderant structural feature of these blocks.

Action on whole alginatu and M-rich blocks. When the enzyme was allowed to act to completion on a number of whole al&rates and M-rich fractions from alginate, the products included some unsaturated oligosaeeharides, but larger fragments were also present. In view of the complete degradation of both poly-G blocks and poly-MG blocks by the enzyme, it seemed probable that these larger fragments were intact poly-M blocks. Gel fiitration of the products on Sephadex G-25 separated a fraction, which was excluded by the gel, from unsaturated oligosaccharides. P.c. of the oligo- saccharides indicated components having the mobilities of d-G, &G-G, and at least one other component. Gel filtration on columns of Sephadex G-100 was used to estimate the chain lengths of these poly-M blocks (Table III). The columns were

ALGINIC ACID i91

calibrated with Dextrans T-10 and T-20, with long-chain poly-G blocks (cl., 80) and with short-chain poly-M (cl., 25).

TABLE III

CHAIN LENGTHS OF POLY-M BLOCKS LEFr AFIER ENZYMIC DEGRADATION OF ALGINATE SAMPLE5

Source of aIginate Chain length (uranic acid residues)

Commercial sample I Commercial sample II A. nodosum (acid-purified)” A. nodosum (Ca2+ complex)o A. nodosum growing tips A. nodosum fruiting bodies Poly-M blocks

25 23 23 24 21-30

“Samples purified in dEerent ways, see text.

It should be noted that the estimated chain-lengths are not considered absolute, but are strictly relative to one another, and that the values obtained are nearer number-average than weight-average, as the method of detection was by absorption at 230 mn, which gives a measure of the unsaturated residue at the end of each chain.

In addition to two commercial alginate samples (I and JLI in Table III), alginates extracted from mature AscupizyZZum nodusum and purified either by acid precipitation or by formation of the calcium complex were examined. Three samples rich in mannuronic acid were examined, one from growing tips of A. nodoszan13, which contained 75% of mannuronate, another from fruiting bodies of A. nodosum’ 3,

which contained 90% of mannuronate, and a sample of poly-M blocks. Table III shows the remarkable uniformity in the lengths of the poly-M blocks left after enzymic degradation of the other blocks. Only in the case of alginate from growing tips of A. nodosum was the elution profile from the column broader than normal, showing greater polydispersity in this fraction. This uniformity in chain lengths of the poly-M blocks suggests a biological control of the length of such blocks. It is known that brown algae contain an epimerase that can convert M-residues into G-residues within the polymeric chain14, and it is tempting to suggest that, during biosynthesis of al&ate in algae, this enzyme comes into play when the polyiM blocks that are being biosynthesized exceed a critical chain-length, estimated by this study to be -24 units.

EXPERIMENTAL

General methods. - The methods of t.l.c., p.c., and paper electrophoresis in borate buffer have been described’*“. The detection of unsaturated oligouronic acids by absorption at 230 run or with the thiobarbituric acid reagent*, of total uranic acids’, and of neutral sugars with the phenol-sulphuric acid reagent i ’ have been discussed previously5. The procedure for converting uranic acids into aldoses within

192 J. BOYD, 3. R. TTJRVEY

oligosaccharides by lactonisation and reduction of the lactones with sodium boro- hydride, followed by acid hydrolysis, has also been described’. Oligosaccharide fractions from columns were desalted either on a column5 of Sephadex G-10 or, for the unsaturated disaccharide, on charcoal-Celiter6.

Enzyme and substrates. - Preparation of the enzyme from KlebsieHa aerogenes

and determination of its activity have been described ‘. Also described previously are the preparation of polymeric blocks from alginate, of the unsaturated tetrasaccharide d-G-G-G’, and of the saturated oligoguluronic acids”‘. Commercial alginate samples I and II are those described as Samples IV and VII in Ref. 17. Alginate was extracted from whole Ascophyihm nodosum (collected in July 1974) by stirring the fronds in 0.2~ HCl (5 vol.) for 6 h. The insoluble material was then macerated in 0.2~ HCl and stirred overnight. The insoluble material was then extracted with aqueous 3% sodium carbonate for 6 h, and the filtered extract poured into ethanol (3 vol.). The precipitate was collected, and redissolved in water, and the solution was dialysed for 48 h and freeze-dried. The crude alginate was purified either by acid precipitation1 3 at pH 2.1 or via the calcium complex”. Alginates from growing tips

of A. nodosztm (collected in July 1974) and from the intercellular substance of fruiting bodies (collected in early April 1974) were extracted as described before13.

Action of the enzyme on oligosaccharides. - Each of the oligosaccharides G-G, G-G-G, G-G-G-G, and d-G-G-G (10 mg) was separately dissolved in water (1 ml) and incubated at 36” with 0.2hi phosphate buffer (pH 7.0, 1 ml) and enzyme solution (1 ml, 40 units) in thermostatted cuvettes (1 cm) in a U&am SP800 spectro- photometer. The absorbance at 230 nm was plotted for 2 h. The oligosaccharides G-G, G-G-G, and d-G-G-G showed no observable increase, whereas G-G-G-G showed an increase of 0.025 durin g the first 6 min; the absorbance thereafter remained constant. Toly-G blocks incubated under the same conditions showed increases of 0.107 (6 min), 0.272 (16 min), and 1.050 (1 h).

Solutions in 0.05M phosphate buKer (pH 7.0) of trisaccharide G-G-G (1.1 mg

per mlj and poly-G blocks (0.24 mg per ml) were mixed in various proportions with

enzyme (40 units) in a total volume of 3 ml. The absorbance at 230 nm was plotted as above. The results are given in Table I.

Acrion on poly-MG blocks. - Poly-MG blocks (150 mg) and sodium chloride (0.5 g) in 0.05~ phosphate buffer (pH 7.0, 75 ml) were incubated at 36” with enzyme (900 units) under a layer of toluene. The absorbance at 230 nm was measured on samples (0.2 ml) diluted to 5 ml, until it remained constant (- 76 h), and the digest was then freeze-dried. The products were desalted, and then fractionated on Biogel P-4 as described previously’. The void volume of the column was 150 ml, and four fractions were eluted: A (268-294 ml), B (296-320 ml), C (322-344 ml), and D (350-386 ml). Fractions A, B, and C were desalted on Sephadex G-10, and the four fractions were examined by p-c. Fractions A (R,,, 0.02), B (O-06), and C (0.17)

appeared to be homogeneous, but fraction D separated into two components, Dr (RoLCA 0.52) and D, (0.31). Fraction D was applied to a column (185 x 1.9 cm) of Sephadex G-10 (swollen in distilled water), and the column was eluted with distilled

ALGINIC ACID 193

water; fractions were monitored by absorption at 230 nm and by the periodate- thiobarbituric acid method. A disaccharide (02) eluted at 400-500 ml did not absorb at 230 nm, but was detected by the other method. Disaccharide D, (540-610 ml) was eluted with salts and was detected by both methods. The solution was concentrated, and desalted on charcoal-Celite. P.c. indicated that D, was pure, but that D, con- tained a trace of D1. Each oligosaccharide was examined by the periodate-thio- barbituric acid method and, with the exception of D,, each gave the chromophore absorbing at 550 nm. D, gave a slight absorbance at 550 nm (G.O6), compared with an

equal weight of D, which gave an absorbance of 1.07. Oligosaccharides C, D, , and II, were each lactonised, reduced with sodium borohydride, and hydrolysed with acid. The products were examined by paper electrophoresis. Fraction C was also examined by ’ 3C-n.m.r. spectroscopy lo.

Enzynzic degradation of alginates. - The alginate (50 mg) and NaCl(G.2 g) were incubated at 35” in 25mM phosphate buffer (pH 7.0, 20 ml) with enzyme (200 units) under a layer of toluene. After 24 h, more enzyme (400 units) was added. Samples (0.5 ml) were withdrawn at intervals, diluted with water (5.5 ml), and monitored at

230 nm. When the absorption was constant (-48 h), showing that enzyme action had ceased, the digest was concentrated at 30” to 2 ml. A small sample was examined by p.c., and the remainder was loaded on a column (2 x 70 cm) of Sephadex G-25, previously equilibrated with aqueous 1% NaCI. Elution was continued with this solvent, and fractions (5 ml) were collected and monitored at 230 run. On a typical column, material eluted near the void volume of the column (130 ml) was clearly separated from oligosaccharides (210-320 ml). The excluded material (poly-M blocks) was freeze-dried.

A portion (-25%) of the foregoing product was mixed with Procion-orange dextranlg (5 mg; mol. wt., 2 x 106) and D-mannuronic acid (5 mg) in aqueous 1% NaCl (0.5 ml), and applied to a column (1.5 x 97 cm) of Sephadex G-100 (preswollen and packed in aqueous 1% NaCI). The column was eluted with aqueous 1% NaCI, and fractions (2 ml) were collected. Elution of the orange dextran was monitored by absorption at 490 MI, of poly-M by absorption at 230 nm, and of mannuronic acid by the method for total uranic acid. From the elution volumes of the three peaks, the distribution coefficient, K,, was calculatedto. The distribution coefficients of dextran T-10 (mol. wt., 1G4), dextran T-20 (mol. wt., 2 x 104), long-chain poly-G blocks

(chain length, 8G), and poly-M blocks (chain length, 25) were determined on the same column and used to plot a graph of log (chain length) against I&_ From this graph, the chain lengths of the residual poly-M blocks were calculated.

ACKNOWLEDGhlENTS

We thank Dr. D. A. Rees for helpful advice, and S.R.C. and Unilever Research

Ltd. for financial support.

194 J. BOYD, J. R. TURVBY

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